SUMMARY Intestinal barrier function is compromised in enteric and systemic diseases, including infectious enterocolitis, food allergy, celiac disease, graft versus host disease (GvHD), and inflammatory bowel disease (IBD). The co-founders of Thelium Therapeutics discovered the central role of myosin light chain kinase (MLCK) in barrier regulation and demonstrated that targeted intestinal epithelial MLCK inhibition limits experimental IBD and GvHD. Unfortunately, severe toxicities associated with barrier-independent MLCK functions in epithelia and other tissues, e.g., smooth muscle, preclude therapeutic targeting of MLCK enzymatic activity. We recently reported (Graham et al., Nature Medicine, 2019) that a specific MLCK splice variant, MLCK1, is central to barrier regulation and depends on interactions mediated by immunoglobulin-cell adhesion molecule domain 3 (IgCAM3). We solved the IgCAM3 crystal structure, identified a drug binding pocket unique to IgCAM3, and screened a library of ~140,000 drug-like molecules. One compound, Divertin, bound IgCAM3, prevented cytokine-induced MLCK1 recruitment, myosin II regulatory light chain phosphorylation, and barrier dysfunction. Critically, Divertin did not inhibit MLCK enzymatic function, epithelial wound healing, or smooth muscle contraction, and in vivo toxicity studies failed to identify adverse effects. Divertin prevented acute TNF-induced barrier loss in vivo (mice) and ex vivo (human intestinal biopsies) and restored immune-mediated barrier loss in vivo (IL-10 knockout mice). Finally, Divertin delayed onset and prevented progression of experimental immune-mediated (T cell transfer) IBD, as indicated by barrier preservation and restoration, reduced mucosal immune activation, and enhanced survival. Although useful as a tool compound, Divertin lacks characteristics required for a lead compound. This proposal will overcome that obstacle to clinical application of Divertin by discovering lead compounds with similar activities. Cutting edge, physics-based rational design and molecular dynamic simulation methods has already been used to probe an expansive compound library and identify those with predicted high-affinity binding to the MLCK1 IgCAM3 crystal structure. Those molecules will be subjected to a rank order screening funnel to identify compounds with suitable MLCK1 binding affinities, efficacy in preserving and restoring epithelial barrier function, and absence of enzymatic inhibitory activity. These lead compounds will be suitable for optimization and development to facilitate IND-enabling studies for a first-in-class barrier-restorative therapy to manage gastrointestinal and systemic diseases.